Rho kinase (ROCK) is a kinase found in all eukaryotic cells. It regulates key processes that include cell motility, cell differentiation, cell survival, cell-cell junctions and expression of extracellular matrix proteins. There are two isoforms of ROCK, ROCK1 and ROCK2. ROCK2 is more highly expressed in the CNS. It is also the form most highly expressed in tissues that have dysregulated ROCK in disease. Therefore ROCK-2 selective inhibitors may be used to treat a variety of diseases accompanied by abnormal or pathological activation of ROCK signaling for instance inflammatory stimuli, the microbiome or other factors that increase the activity of ROCK2 leading to progression of disease.
The Abl tyrosine kinase was identified as a critical driver of leukemia from studies of the Abelson murine lymphosarcoma virus that induced cellular transformation and lymphomas. Subsequent studies demonstrated that chromosomal translocation of ABL1 to the breakpoint cluster region (BCR) gene sequences results in production of the BCR-ABL1 fusion protein and elevated tyrosine kinase activity in patients with Philadelphia (Ph) chromosome-positive human leukemia. Subsequently studies of Abl show that, like ROCK, it regulates many cellular processes leading to disease. Abl is not typically active in neurons, but is activated in many neurological diseases. Abl regulates diverse cellular processes and can be activated by multiple stimuli leading to cytoskeletal reorganization and cell survival. There are two isoforms of Abl: Abl1 and Abl2. Abl1 is the form of interest for this application. It is sometimes called c-Abl or Abl in the literature, and we refer to it as Abl.
An off-target activity of a ROCK2 inhibitor on Abl kinase may be of benefit in treating ophthalmological diseases of retinal ganglion cells. ROCK2 and Abl are key kinases that regulate homeostatic balance of the cytoskeleton, and their perturbation and kinase hyper-activation causes neuronal dysfunction and cell death. The neuronal cytoskeleton of projection neurons such as retinal ganglion cells is particularly susceptible to disturbances in cytoskeletal regulation because of the long axonal process and requirement for axonal transport. If a retinal ganglion cells was the size of a Volkswagen Beetle, its axon would be 2 miles long.
Ophthalmology
Glaucoma is a disease that affects retinal ganglion cells (RGCs), and changes at the optic nerve head where the RGC axons exit the retina are one of the first visual hallmarks of disease (Quigley. 2016 Annu Rev Vis Sci. 2: p. 235-254). It has been estimated that glaucoma will affect more than 80 million individuals worldwide by 2020, with 6-8 million individuals becoming bilaterally blind. Glaucoma is the second leading cause of irreversible blindness, one of the most prevalent neurodegenerative diseases. Glaucoma starts with a loss of peripheral vision and painlessly progresses slowly, eventually leading to vision loss, then blindness. Visual loss results from loss of RGCs, and that reduced aqueous humor drainage through the trabecular meshwork (TM) and Schlemm's canal is the root cause of ocular hypertension in glaucoma. In the initial stages, activities involving glare and dark adaption are affected which impacts driving and mobility; motor accidents and falls are early consequences of glaucoma. The total annual economic impact of visual disorders to the healthcare system for Americans aged 40 years and older is estimated at $35 billion.
Many forms of glaucoma are associated with elevated intraocular pressure (IOP) and standard treatment is to reduce IOP with drugs. Because progression of glaucoma is slow and painless, noncompliance for daily use of IOP-reducing medications is high. Side-effects make non-compliance even more likely because there is no immediate impact when eye drops are not applied. Even with daily treatments, some patients show continuous progression of glaucoma despite reaching their lowest achievable IOP (Chang et al. 2012 Ophthal. 119(5): p. 978-986). Failure to keep IOP reduced results in irreversible damage, and patients do not lose vision until there is permanent neuronal loss. Lowering intraocular pressure slows the progression of disease, but lowering IOP does not address the underlying mechanism of RGC death and optic nerve degeneration. Therefore, glaucoma is controlled, but never cured by daily use of available eye drops that reduce IOP.
There are six classes of topical ocular hypotensive drugs used to lower IOP. Prostaglandin analogs are the biologically active metabolites of arachidonic acid and its analogs that are commonly used to reduce IOP. They may reduce IOP by 27%-33%, typically require once daily dosing, and are generally associated with good compliance. Rho kinase (ROCK) inhibitors have potential to slow blockage of the TM by reducing fibrosis, thereby slowing RGC death. However, non-specific ROCK inhibitors in clinical development cause significant hyperemia, a side effect that leads to non-compliance, although long-term use would be needed to effectively slow disease progression. Non-specific ROCK inhibitors have been shown to be neuroprotective, but only by intravitreal injection (Kitaoka et al. 2014 Brain Res. 1018(1): p. 111-118), which is not a feasible delivery for repetitive treatment in humans. Thus, drugs that reduce IOP and slow disease progression are urgently needed to prevent blindness in glaucoma.
In the eye, the TM is a mechanosensitive structure that regulates aqueous humor outflow. Aqueous humor is produced by the ciliary body epithelium lining, and it drains out of the eye through the TM into Schlemm's canal and into the episcleral venous system. Glaucoma is believed to be associated with changes in the TM that increase deposition of extracellular matrix (ECM) adjacent to Schlemm's canal (Tektas et al. 2009 Exp Eye Res. 88(4): p. 769-775), a process regulated by ROCK (Pattabiraman, P. P. et al., 2016, Eur. J. of Pharm., 787: P. 32-42). Hyperactivation of ROCK may increase deposition ECM in human TM cells, slowing drainage (Pattabiraman et al. 2014 J Cell Physio. 229(7): p. 927-942). Thus, ROCK inhibitors that suppress fibrogenic activity of TM cells would loosen the TM to increase aqueous humor outflow and reduce IOP.
There are two forms of ROCK that may be implicated in glaucoma. The TM has both ROCK1 and ROCK2 and RGCs have more ROCK2. ROCK2 is more important for RGC regeneration (U.S. Pat. No. 7,572,913., 2009). Y-27632 and Fasudil, targeting both ROCKs are the most widely used reference ROCK inhibitors for research. There have been 7 different ROCK inhibitors tested in human clinical trials, most with equal affinity to ROCK1 and ROCK2 (Ren et al. 2016 Invest ophthal Vis Sci. 57(14):p. 6197-6209). Lack of therapeutic window has hampered the development of ROCK inhibitors, even when used topically to treat eye diseases (Defert et al. 2017 Expert Opin Ther Pat. 27:507-515).
ROCK inhibitors may reduce IOP by increasing aqueous humor outflow through the TM, by contrast to available IOP-reducing drugs that act on the unconventional pathway of uveoscleral drainage (Whitlock et al. 2009 J Ocul Pharmacol Ther. 25(3): p. 187-194). Rho/ROCK pathway is often activated in disease, and they also have potential to be neuroprotective and increase plasticity and regeneration of RGC injury. Netarsudil (previously AR-33324) is the only ROCK inhibitor approved in the USA. Ripasudil is approved in Japan, but not the USA. Both inhibitors cause hyperemia (red eyes) as a major side effect (Bacharach et al. 2015 Ophthalmology 122(2): p. 302-307., Tanihara H. et al. 2016 Acta ophthalmol. 94(1): p. e26-e34), and therefore patient compliance is expected to be problematic.
There is a need for ROCK inhibitors causing reduced or no hyperemia. There is a need for newdisease-modifying treatments for glaucoma.
Retinitis pigmentosa (RP) is a degenerative retinal dystrophy caused by the progressive degeneration of the rod photoreceptor cells in the retina. This form of retinal dystrophy manifests initial symptoms independent of age. The progressive rod degeneration is followed by abnormalities in the adjacent retinal pigment epithelium (RPE) and the deterioration of cone photoreceptor cells. As peripheral vision becomes increasingly compromised, patients experience progressive “tunnel vision” and eventual blindness. Affected individuals may additionally experience defective light-dark adaptations, nyctalopia (night blindness), and the accumulation of bone spicules in the fundus. RP is relatively rare inherited disorder that results from mutations in any one of more than 50 genes required for making proteins that are needed in functioning photoreceptor cells.
Macular degeneration, also known as age-related macular degeneration (AMD or ARMD), is an eye disorder affecting over 235 million people world-wide. Macular degeneration results in blurred or no vision in the center of the visual field, but does not result in complete blindness. Visual hallucinations may also occur but these do not represent a mental illness. Macular degeneration is the result of damage to the macula of the retina. It may be age-related, but genetic factors and smoking also play a role. The severity is divided into early, intermediate, and late types, with the late type being further divided into “dry” and “wet” forms. The dry form makes up 90% of cases. Supplements in those who already have the disease may slow progression, but there is no cure or treatment that returns vision already lost. In the wet form, anti-VEGF medication injected into the eye or less commonly laser coagulation or photodynamic therapy may slow worsening. Targeting VEGF may reduce pathological growth of blood vessels in the retina that contribute to pathology of disease.
There is a need for new therapies for retinitis pigmentosa, macular degeneration, and retinal angiogenesis.
Diseases affecting the cornea are a major cause of blindness worldwide, second only to cataract in overall importance. The epidemiology of corneal blindness is complicated and encompasses a wide variety of infectious and inflammatory eye diseases that cause corneal opacity and scarring, which ultimately leads to functional blindness. There have been a number of studies that indicate potential usefulness of ROCK inhibitors for treatment of corneal diseases that include Fuchs' corneal dystrophy, corneal scarring, and prevention of scaring complication in glaucoma surgery.
Fuchs' corneal dystrophy is a progressive, hereditary disease of the cornea which is late onset and slowly progressing. Patients often present in the fifth to sixth decade of life with blurry morning vision that increases in duration as the disease progresses. Symptoms at presentation include painless decrease in visual acuity, photophobia, glare and halos around lights. It is a condition of the posterior cornea and characteristic features include the formation of focal excrescences of Descemet membrane termed ‘guttae’, and loss of endothelial cell density. As disease advances, corneal edema results in the development of painful subepithelial and epithelial bullae, and may progress to loss of corneal sensation, visual acuity and, ultimately, the development of corneal opacification and pannus formation. The ROCK inhibitor Y27432 has been used to treat patients with Fuchs membrane dystrophy. Upon treatment corneal clarity improved and vision improved for the 24 months the patient was followed (Norika et al 2013. Cornea 32:1167-1170). ROCK inhibitors inhibit keratocyte-to-myofibroblast transition, and topical application after a superficial lamellar keratectomy elicits an altered wound healing response, with evidence of an embryonic-type deposition of collagen fibrils thus avoiding scar tissue formation in preference to an ordered regeneration of the wounded tissue (Yamamoto 2012. Mol Vis. 18:1727-1739).
In the surgical treatment for glaucoma, the most common complication of glaucoma surgery is scar formation induced by activation of a wound healing response that causes fibrosis at the surgical site. Rho kinase inhibitors reduce activation of human conjunctival fibroblasts and that treatment with Rho kinase inhibitor via eyedrops significantly suppresses scar formation (Futakuchi et al. 2016. Experimental eye research. 149:107-115). Similarily, BA-1076 will be of therapeutic use in preventing excessive scarring after glaucoma filtration surgery.
There is a need for new therapies for the treatment of corneal blindness, Fuchs' corneal dystrophy, and corneal scarring, and for reducing post-operative scarring (e.g., post-glaucoma surgery corneal scarring).
Gastrointestinal Disorders
Tight junctions are crucial determinants of barrier function in polarized intestinal epithelia and are significantly regulated by activity of the Rho-ROCK pathway (Walsh et al., Gastroenterology, 2001; 121(3):566). Many conditions can impact negatively on barrier function in the intestinal epithelium ranging from inflammation to radiation exposure. It is also known that inhibition of the Rho-ROCK pathway can limit the activation of pro-fibrotic pathways, such as are activated in the setting of inflammatory bowel disorders, and positively impact on paracellular permeability through tight junctions (Du et al., Gastroent. Res. Pract.; 2016; 2016: 7374197). Importantly, evidence has also suggested that inhibition of the c-Abl signaling pathway may also show anti-fibrotic effects. Having an inhibitor targeting both ROCK and c-Abl may provide a novel therapeutic approach in this setting.
Ionizing radiation can be emitted from atoms of radioactive isotopes and can be released accidently (e.g., nuclear accident), by medical procedure (e.g., radiation treatment of cancer) or by bombs during war. Radiation is a high-energy particle or electromagnetic radiation that deposits energy when it interacts with atoms, resulting in ionization (electron excitation). As a result, an affected cell may either die or malfunction. The radiation can damage a cell directly by DNA damage, or indirectly through the creation of unstable, toxic hyperoxide molecules; which in turn can damage sensitive molecules and afflict subcellular structures. Radiation damage primarily affect proliferating cells, and the cell intestine has a very low threshold to radiation damage because of fast cell turnover. Bone marrow tissue is also sensitive. Symptoms of acute radiation poisoning are dependent on the absorbed dose, with symptoms appearing hours to days. There are treatments for the hematologic disorders that follow radiation poisoning (e.g., bone marrow transplants, and treatment with G-CSF (Neupogen). There are no effective treatments for the gastrointestinal (GI) disorders in ARS.
The polarized cells epithelial cells of the GI tract that form a protective barrier against commensal and pathogenic microorganisms play an important barrier function, in addition to their role in regulating absorption of nutrients, water, and ion homeostatic. GI-acute radiation syndrome (ARS) the destruction of the intestinal epithelial lining causes breakdown of the mucosal barrier, resulting in diarrhea, dehydration and electrolyte imbalance. Although all cellular compartments may contribute to and modulate organ dysfunction, the key event in the pathophysiology of intestinal radiation toxicity is enterocyte depletion, with possible vascular damage contributing at higher radiation doses. IN GI-ARS there is loss of intestinal clonic cells, leading to loss of epithelia crypts. The severity of mucosal breakdown is dose dependent, and occurs at radiation levels higher than those that destroy bone marrow. In the highly polarized epithelial cells of the GI tract, maintaining the correct balance of active and inactive ROCK is critical to function of the tissue. Over activation of Rho cause loss of barrier function because it is a key regulator of adherens and tight junctions. The ROCK pathway has been identified as a target of for modulation of intestinal radiation-induced toxicity (Haydont et al, British Journal of Radiology, 80 (2007), S32-S40).